PROJECT SUMMARY/ABSTRACT Lung cancer is currently the leading cause of death worldwide. The majority of lung cancer patients are classified as having non-small cell lung cancer (NSCLC) histologically with greater than 50% of patients presenting with metastasis. Additionally, the overall 5-year survival rate is only 16% highlighting the critical need for improved therapies through innovative research. To date lung cancer research and therapies focus almost exclusively on protein coding genes thereby missing the potentially promising class of long noncoding RNAs (lncRNAs). To address this, we leveraged recent advances in sequencing technologies to perform the first ab initio transcriptome assembly from ~550 lung cancer patients to discover 111 Lung Cancer Associated Long non-coding RNAs (LCALs) altered in lung cancer. However, our current understanding of how lncRNAs function in cancer is still in its infancy. Several examples indicate that lncRNAs may be master regulators in cancer biology, typically binding with chromatin modifying complexes, such as the Polycomb Repressive Complex 2, and guiding these complexes to genomic regions to regulate gene expression. Supporting this, we have discovered and characterized a novel lncRNA, LCAL5, that is overexpressed in lung cancer, promotes aggressive phenotypes, interacts with the protein complex, Polycomb Repressive Complex 2 (PRC2), and associates with poor patient survival. Moreover, while the majority of lncRNAs show tissue- and lineage- specific expression, transcriptome analysis of our compendia of ~8,000 patients across cancer types revealed that LCAL5 is among a small subset of lncRNAs altered in multiple solid tumors suggesting its conserved and critical oncogenic role. Therefore, we hypothesis that LCAL5 is important in promoting aggressive disease through its interaction with, and recruitment of PRC2 to chromatin, to epigenetically modify genes associated with metastasis. To accomplish this, Aim 1 will determine the specific EZH2 interaction sites within LCAL5 necessary to epigenetically regulate genes associated with metastasis and promote oncogenic phenotypes. Generation of LCAL5 deletion constructs will determine the interaction sites needed for EZH2 binding and function. The deletion construct lacking this interaction site will be used to determine how changes in LCAL5 scaffolding with EZH2 modulates PRC2 occupancy (ChIP-Seq) and alters gene expression (RNA-Seq) in comparison to overexpressing the full length LCAL5. Further, Aim 2 will determine that LCAL5 promotes metastasis in vivo through generation of a CRISPR/Cas9 LCAL5-knockout cell line. This study will have a large overall impact on the field of lncRNA tumor biology by providing mechanistic insights into LCAL5- dependent epigenetic regulation necessary to confer aggressive phenotypes. Given the deregulation of LCAL5 in multiple cancers, we believe LCAL5 has a conversed function in promoting tumorigenesis across cancer types thus broadening the impact of this study. Our findings also have potential translational implications for the development of a novel therapeutic target to transform lung cancer treatment.